Nitrile-synthesizing enzyme: Screening, purification and characterization

  • Kumano Takuto
    Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, University of Tsukuba
  • Suzuki Takahisa
    Division of Applied Life Science, Graduate School of Agriculture, Kyoto University
  • Shimizu Sakayu
    Division of Applied Life Science, Graduate School of Agriculture, Kyoto University
  • Kobayashi Michihiko
    Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, University of Tsukuba

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Abstract

Cyanide is known as a toxic compound for almost all living organisms. We have searched for cyanide-resistant bacteria from the soil and stock culture collection of our laboratory, and have found the existence of a lot of microorganisms grown on culture media containing 10 mM potassium cyanide. Almost all of these cyanide-resistant bacteria were found to show β-cyano-L-alanine (β-CNAla) synthetic activity. β-CNAla synthase is known to catalyze nitrile synthesis: the formation of β-CNAla from potassium cyanide and O-acetyl-L-serine or L-cysteine. We found that some microorganisms were able to detoxify cyanide using O-methyl-DL-serine, O-phospho-L-serine and β-chloro-DL-alanine. In addition, we purified β-CNAla synthase from Pseudomonas ovalis No. 111 in nine steps, and characterized the purified enzyme. This enzyme has a molecular mass of 60,000 and appears to consist of two identical subunits. The purified enzyme exhibits a maximum activity at pH 8.5–9.0 at an optimal temperature of 40–50°C. The enzyme is specific for O-acetyl-L-serine and β-chloro-DL-alanine. The Km value for O-acetyl-L-serine is 10.0 mM and Vmax value is 3.57 μmol/min/mg.

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